This invention broadly relates to the field of molecular genetics. More specifically, the invention relates to methods for genetic analysis of DNA sequences corresponding to ancestral haplotypes of multigene complexes such as the Human Major Histocompatibility Complex and fragments thereof.
The Human Major Histocompatibility Complex (MHC) is a region of chromosomal DNA which plays a key role in the immune system and influences diverse functions and diseases. The MHC contains multiple polymorphic and duplicated genes (Zhang et al, 1990). At the centromeric end of the human chromosome on which the MHC is found there are genes such as HLA DR, DQ and DP which encode the Class II products involved in specific antigen presentation. At the telomeric end of the chromosome there are multiple genes.uch as HLA-A, B and C which encode the Class I products recognised by cytotoxic T cells. In the central region there are genes for amplifiers and mediators such as C4, C2, Bf and tumour necrosis factors (TNF-a, TNF-b), together with a heterogeneous collection of at least ten other genes (Sargent et al, 1989; Spies et al, 1989). Although only some of these genes have been sequenced it is already obvious that there is no single gene family, in that the products vary substantially in their structure. Genes such as heat shock protein 70 may possibly have a role in antigen processing prior to presentation. The products of some of the other genes may possibly be involved in intercellular adhesion, and perhaps some could influence immune responses as accessory molecules (Banerji et al, 1990). The B144 gene, together with the complement and TNF genes, could possibly play a role in the regulation of antibody production (French and Dawkins, 1990). Although more detailed information is required, it is possible that the MHC could be thought of as a chromosomal region containing heterogeneous genes which in various ways regulate and direct immunological responses, whether mediated by antibody, T cells or other effector mechanisms.
For many years it has been known that the MHC contains genes which influence the susceptibility to many diseases (Dawkins et al, 1983). Products of the human MHC are also intimately involved in tissue rejection, wherein transplanted tissues bearing MHC products which are non-identical to the transplant recipient are recognised as foreign by the immune system and rejected.
Because of the practical importance of transplantation, much effort has been spent in xe2x80x9ctissue typingxe2x80x9d within the MHC to ascertain whether candidate tissues for transplantation carry the same MHC ancestral haplotype as the intended recipient. Most of this typing has been by serological analysis focusing on the DR and DQ HLA class II gene products, the HLA B class I gene products, as well as other markers.
Current tissue typing techniques are subject to a degree of ambiguity which is inherently associated with serological determinations as well as time delays associated with such analysis. There is additionally a need for further xe2x80x9cmarkersxe2x80x9d in order to assess the compatibility or otherwise of tissue for transplantation.
xe2x80x9cTissue typingxe2x80x9d is also important in assessing susceptibility and resistance to disease as mentioned above. Where a patient""s susceptibility to autoimmune or other diseases can be detected at an early stage, appropriate therapeutic regimes, and counselling can be implemented before disease progression with attendant advantages.
Ancestral haplotypes are DNA sequences from multigene complexes such as MHC. The ancestral haplotypes of the MHC extend from HLA B to HLA DR and have been conserved en bloc. These ancestral haplotypes and recombinants between any two of them account for about 73% of ancestral haplotypes in our caucasian population. The existence of ancestral haplotypes implies conservation of large chromosomal segments. These ancestral haplotypes carry many MHC genes, other than the HLA, which may be relevant to antigen presentation, autoimmune responses and transplantation rejection. Tissue typing is an analysis of the combination of alleles encoded within the MHC. Many of these allelic combinations can be recognised as ancestral haplotypes. Other multigene complexes containing ancestral haplotypes include the lipoprotein gene complex and the RCA complex
This invention stems from characterisation of DNA corresponding to ancestral haplotypes. The inventors have surprisingly found that the DNA sequences corresponding to a particular ancestral haplotype are identical within that ancestral haplotype, whereas DNA sequences between ancestral haplotypes are very polymorphic. Each ancestral haplotype, therefore, possesses a unique nucleotide sequence which can be exploited in assigning ancestral haplotypes. Accordingly, genetic analysis and assignment of ancestral haplotype maybe readily conducted according to the methods of this invention. This is particularly important where identity at the genomic sequence level may be necessary for the most desirable outcome following grafts and transplants such as in bone marrow grafting (Christiansen et al, 1991). The genetic analysis of the present invention provides the ability to xe2x80x9cmatchxe2x80x9d ancestral haplotypes between individuals or to xe2x80x9ctypexe2x80x9d ancestral haplotypes by a comparison to a reference panel of ancestral haplotypes.
In accordance with the first aspect of this invention, there is provided a method for genetic analysis comprising comparing a first DNA sequence corresponding to an ancestral haplotype or recombinant thereof of a multigene complex, such as the MHC, with one or more reference DNA sequences each corresponding to a separate ancestral haplotype so as to establish identity or non-identity there between and thus an assignment of ancestral haplotype of said first DNA sequence.
DNA sequences which may be compared in assigning ancestral haplotype may comprise the DNA sequence of any multigene complex such as but not limited to the lipoprotein gene complex, the RCA complex and the MHC complex. Preferably the multigene complex is the MHC such as HLA C between HLA B and TNF+B144, and/or a DNA sequence corresponding to one or more haplospecific geometric elements of the human Major Histocompatibility Complex as defined hereinafter.
The present invention is directed to genetic analysis of the genomes of higher organisms such as from mammals, plants and insects. Preferred mammals are humans, livestock animals, companion animals and wild animals. Most preferably, the mammal is a human.
DNA sequences corresponding to ancestral haplotypes or recombinants thereof may be readily analysed by a number of techniques such as DNA sequence analysis, restriction fragment length polymorphism (RFLP), reaction with haplospecific oligonucleotide probes, heteroduplex analysis, primer directed amplification and other methods as are well known in the art. The genome itself may be subject to the analysis or via cDNA or mRNA.
In a preferred aspect of the present invention, the comparison is by the use of oligonucleotide probes which may also be labelled with a reporter molecule or a primer to direct amplification.
According to this preferred aspect, there is provided a method for matching a particular ancestral haplotype in the genome of two or more individuals of a higher organism, said method comprising contacting a region of the genome or fragment or portion thereof from each individual with an oligonucleotide probe which hybridises to at least one complementary sequence within the ancestral haplotype and comparing the extent of hybridisation to match the non-identity or identity of the ancestral haplotype.
Another preferred aspect of the present invention provides a method for matching a particular ancestral haplotype in the genome of two or more individuals of a higher organism, said method comprising contacting a region of the genome or fragment or portion thereof from each individual with at least one oligonucleotide primer which hybridises to at least one complementary sequence within the ancestral haplotype and comparing the profile of hybridisation or genomic amplification products, or nucleotide sequence of such amplification products to match the non-identity or identity of the ancestral haplotype.
Yet another preferred aspect of the present invention provides a method wherein the oligonucleotide probe hybridises to multiple complementary sequences within the ancestral haplotype.
A DNA sample may be isolated from an individual and then subject to characterisation as detailed above to assign ancestral haplotype. For example, a DNA sample may be isolated and analysed for specific hybridisation with an ancestral haplotype specific probe as provided according to this invention.
Alternatively, ancestral haplotype sequences within a DNA sample may be amplified, for example, using conventional techniques such as linear amplification using a single primer which hybridises to ancestral haplotype sequences, or by amplification using paired primers. Amplified sequences may then be detected directly by visual analysis of separated fragments or by reaction with ancestral haplotype specific or non-ancestral haplotype specific probes.
By ways of further example, a DNA sample from an individual may be reacted with one or more restriction endonucleases, the resultant fragments separated, for example by gel electrophoresis, followed by subsequent analysis of restriction fragments using a relevant MHC specific probe by Southern analysis (Sambrook et al., 1989). Resulting restriction fragment patterns may then be compared with restriction fragment patterns prepared from reference samples of known ancestral haplotypes. By a comparison of restriction fragment polymorphisms with reference samples, a designation of ancestral haplotype may be made. This is possible by virtue of the absolute conservation of ancestral haplotype sequences within a specific ancestral haplotype.
By a comparison of ancestral haplotype nucleotide sequences, the inventors have surprisingly identified polymorphic regions within the ancestral haplotype which comprises stable stretches of nucleotides which differ between ancestral haplotype. Preferably, these polymorphic regions are haplospecific geometric elements (HGEs).
There are approximately 50 ancestral haplotypes selected in the human caucasoid population, with each ancestral haplotype possessing Haplospecific Geometric Elements. The HGEs are geometric in that there is a mathematical relationship between the number of bases which is a characteristic of each ancestral haplotype. There is also geometry in the sense that there is a symmetry around the center of the region which is defined from the boundaries which are more or less common to different ancestral haplotypes. HGEs are also distinctive in that there is non-random usage of nucleotides with iteration of certain components of the sequence. While these components may contain simple sets (eg di and trinucleotide iterations), these do not themselves define the elements and do not allow recognition of haplospecificity or geometric patterns.
As will be described hereinafter, HGEs have been shown to occur at various sites within the MHC. Elements at each of these sites may be related to each other in that they have the same or predictable geometry.
It should be appreciated that the detection of HGEs, and indeed the characterisation of DNA sequences corresponding to ancestral haplotypes or recombinants thereof are not dependent upon the use of any specific technique. As described herein, a variety of techniques can be used for identification and characterisation of ancestral haplotype specific sequence sequences.
While HGEs are characteristic of each individual ancestral haplotype, and characterisation thereof therefore provides direct information as to ancestral haplotype, nucleotide sequences outside of the HGEs may also be utilised to distinguish between ancestral haplotypes. The inventors have discovered that ancestral haplotype sequences differ from one another along their length notwithstanding that marked variation occurs within HGEs. Accordingly, the nucleotide sequence of different ancestral haplotypes may be ascertained and the respective differences thereberween used to construct polynucleotide probes which discriminate between ancestral haplotypes. Preferably, the probes hybridize to complementary sequences in a region flanking the HGE and will hybridize to complementary sites represented at least twice.
In accordance with an aspect of this invention, there is provided a method for genetic analysis which comprises the steps of:
(a) hybridising one or more polynucleotide primers having complementary nucleotide sequence to nucleotide sequences flanking HGEs with a sample containing a DNA sequence corresponding to a multigene complex such as the MHC;
(b) amplifying HGEs within multigene complex by multiple cycles of primer extensions; and
(c) detecting the amplified products resulting from primer extension of the HGEs, which products are characteristic of ancestral haplotypes or recombinants thereof.
Single primer sequences may be utilised for amplification (such as linear amplification) whereafter amplified products may be detected by hybridisation with probes complementary in sequence to said amplified HGE.
Paired nucleotide sequences flanking HGEs may be used to amplify the HGEs following multiple cycles of primer extension. Amplified products may be detected by direct visual analysis after fractionation on a gel or other separation medium.
HGEs, or indeed other regions of the ancestral haplotype of the human MHC may be amplified by direct amplification of single stranded RNA or denatured double stranded DNA. Such methods, which employ T7 RNA polymerase to produce large numbers of copies from each template molecule are described by Compton, 1991.
In accordance with a further aspect of this invention, there is provided a method for the detection of ancestral haplotype which method comprises the steps of:
(a) comparing nucleotide sequences of one or more ancestral haplotypes to ascertain polymorphisms between said ancestral haplotype sequences;
(b) constructing polynucleotides from any sequence region between ancestral haplotype sequences which will discriminate between different ancestral haplotypes;
(c) utilising said polynucleotides of step (b) to detect an ancestral haplotype of a multigene complex such as the MHC from the genome of a higher organism sample by hybridising said polynucleotide to said genome and thereafter detecting polynucleotide binding or absence thereof.
In this method, polynucleotides may be used to amplify selected sequences of ancestral haplotypes, the production of amplified sequences corresponding to ancestral haplotype identification.
The aforementioned HGEs have particular utility as surrogate markers for HLA B. HGEs are proximal to HLA B on the MHC lying some 30 to 50 kilobases towards the centromeric end of human chromosome 6. Accordingly, characterising HGEs allows a direct inference as to the ancestral haplotype at the HLA-B locus. Given that recombination in between HGEs and the HLA B would be expected to be a rare event, the assignment of an ancestral haplotype based on characterisation of the HGE, particularly within the CL-1 locus, should hold true for the HLA Ballele, except for the situation where recombination takes place between the HGEs and the HLA B allele.
In a preferred aspect of the present invention, there is provided a method for identifying a ancestral haplotype in the genome of an individual of a higher organism, said method comprising amplifying the ancestral haplotype or portions thereof using a primer capable of hybridising to complementary sequences represented at least once within the ancestral haplotype and comparing the resultant amplication products with a reference of amplication products from a known ancestral haplotype using substantially the same primer to thereby establish identity or non-identity therebetween.
In accordance with a further aspect of this invention, there is provided a method for surrogate typing at the HLA B allele, which method comprises characterising the nucleotide sequence of a HGE.
The nucleotide sequence of HGEs may be carried out by methods well known in the art for the characterisation of any nucleotide sequences.
In accordance with a still further aspect of this invention, there is provided a method for genetic analysis which comprises the steps of:
(a) digesting a DNA sample corresponding to a first human ancestral haplotype or a recombinant thereof with one or more restriction endonucleases, which restriction endonucleases do not cleave within the HGEs of a multigene complex such as the MHC;
(b) separating and analysing the DNA restriction fragments so produced, and comparing the same with one or more reference samples comprising ancestral haplotypes of known ancestral haplotype or recombinants thereof cleaved with said one or more restriction endonucleases of step (a) so as to establish identity or non-identity therebetween.
In accordance with yet another aspect of this invention, there is provided genomic DNA corresponding to an ancestral haplotype of a multigene complex such as the MHC or a fragment thereof. By way of exanple, there is provided genomic DNA corresponding to ancestral haplotypes 57.1, 7.1, 8.1, 18.2, 46.1 and 62.1 of the MHC. These ancestral haplotypes are representative of approximately 50 ancestral haplotypes of which about 22 represent approximately 72% of ancestral haplotypes of the human MHC in a caucasoid population. Following the methods of this invention, any ancestral haplotype genomic DNA or fragment thereof may be isolated utilising hybridisation probes which recognise common sequences between ancestral haplotypes. For example, such conserved sequences flank HGEs.
This invention provides in a particular aspect a genomic DNA sequence corresponding to a fragment of ancestral haplotype 57.1 having a nucleotide sequence as set forth in FIG. 1. This invention also includes unique fragments of such sequence which would generally comprise in excess of 30 to 50 nucleotides. It is to be appreciated that searches can be readily conducted through DNA data bases to establish uniqueness of fragments which are characteristic of ancestral haplotypes. This aspect of the invention further extends to protein products encoded by one or more genes encoded by said nucleotide sequence. Protein products encoded by said one or more genes or fragments thereof may have particular utility as therapeutic immunoregulatory agents, and are included within the scope of the present invention.
In another aspect this invention relates to a HGE of a multigene complex such as the MHC.
HGEs of characteristic nucleotide sequence are carried by each ancestral haplotype. As a consequence, HGEs are characteristic of each ancestral haplotype of, for example, the MHC. As previously mentioned, HGEs possess geometry in the sense that there is a symmetry around the centre of the region which is defined from the boundaries which are more or less common to different ancestral haplotypes. HGEs are also distinctive in that there is non-random usage of nucleotides with iteration of certain components of the sequence, namely di and trinucleotide iterations.
HGEs are characterised by possessing conserved sequences at their boundaries and a variant number of di and trinucleotide repeats in the central region.
Examples of HGEs have the following sequences:
or polynucleotide comprising said nucleotide sequence, or a fragment or derivative thereof capable of hybridising to sequences flanking the HGEs of the MHC.
Preferred primers of the present invention are those set forth below in the 5xe2x80x2 to 3xe2x80x2 direction:
In still another aspect of this invention, there is provided a recombinant vector comprising a human ancestral haplotype, recombinant thereof or fragment thereof, such as a HGE. Recombinant vectors may comprise plasmids, bacteriophage sequences or any other DNA and/or RNA construct as are well known in the art for the maintenance and replication of nucleotide sequences in a host cell, namely a prokaryotic or eukaryotic host cell.
Recombinant vectors generally comprise a selectable marker, for example one or more genes corresponding to antibiotic or drug resistance, or one or more genes corresponding to one or more factors, such as enzymes, requisite for host cell viability. Recombinant vectors generally further comprise an origin of replication which allows for replication of said vector within a host cell, as well as one or more restriction sites to enable the introduction of desired genes or nucleotide sequences into said vector. Myriad host cells are well known in the art and are described for example in Sambrook et al. (1989).
In one specific embodiment, this invention relates to a YAC vector, as described by which comprises a genomic DNA sequence corresponding to an ancestral haplotype of the MHC, a fragment thereof, or HGE as hereinbefore described.
In still a further aspect of this invention, there is provided a human cell line homozygous for a human ancestral haplotype of the MHC.
Human cells lines homozygous for human ancestral haplotypes of the MHC may comprise immortalised lymphocytes or other immortalised human cell types. Immortalisation may be carried out, for example, by transformation with a virus, such as Epstein Barr Virus (EBV).
In yet another embodiment of the present invention, the identification of an ancestral haplotype can be accomplished by multiple priming using one primer or a set of primers. According to this aspect of the invention, there is provided a method for identifying an ancestral haplotype on the genome of an individual comprising amplifying multiple regions within said haplotype with a single primer or set of primers and comparing the amplification products with a reference panel of ancestral haplotypes or with the amplification products from another individual. Furthermore, multiple priming has been shown in the HLA delta block as shown in FIG. 13.
This invention will now be described, by way of example only, with reference to the following non-limiting Figures and Examples.